(1,3-1,4)-.beta.-glucanases are used in the manufacture of different food products and animal feed and as subsidiary materials in biological research when it is necessary to cleave the .beta.-glycosidic linkages in (1,3-1,4)-.beta.-glucans. Especially in the brewing industry the use of such glucan hydrolyzing enzymes permits the application of larger proportions of raw grain in substitution for the use of malt, without this causing any trouble in the filtration due to high viscosity of the mash which may be caused by an increased amount of glucan compounds.
The mixed linked (1,3-1,4)-.beta.-glucans constitute the major part of the endosperm cell walls of cereals like oat and barley. They may cause severe problems in the brewing industry such as reduced yield of extract and lowered rates of wort separation or beer filtration. Remaining .beta.-glucans in the finished beer may lead to the formation of hazes and gelatinous precipitates (Godfrey, 1983). Barley (1,3-1,4)-.beta.-glucanases (EC 3.2.1.73) are synthesized in the scutellum and the aleurone layer during the early stages of germination of seeds (McFadden et al., 1988). However, a large proportion of the malt .beta.-glucanase is irreversibly heat inactivated during kilning and the remaining activity is rapidly destroyed during mashing (Loi et al., 1987).
It has long been known that the viscosity of the wort can be reduced by using .beta.-glucanases from mesophilic Bacillus strains, e.g. from Bacillus amyloliquefaciens or Bacillus subtilis. A serious disadvantage with the known glucanases is their temperature sensitivity, which implies that they are only effective during the early phase of the mashing process. Later on when temperatures are above 65.degree. C. their activity is reduced substantially.
In an attempt to obtain a more thermostable glucanase, the gene from Bacillus macerans encoding glucanase was introduced into Bacillus subtilis in order to express the gene in this organism (DD Patent Application WP C12N/315 706 1). However, at 70.degree. C. this glucanase is also rapidly and irreversibly denatured. Another drawback to the known glucanases in relation to the brewing process is that these glucanases do not exert their full activity in the pH range from 4 to 5 which is the normal condition during mashing. For example the activity of the Bacillus .beta.-glucanase at pH 4.6 is only 20% of that between 6 and 7. Furthermore, the stability is reduced when the glucanase is incubated at pH 4.
The best characterized bacterial (1,3-1,4)-.beta.-glucanases are those from Bacillus subtilis and B. amyloliquefaciens where the genes encoding the enzymes have been cloned and sequenced (Borriss et al., 1985; Cantwell and McConnell, 1983: Hofemeister et al., 1986; Murphy et al., 1984). It has recently been shown that the .beta.-glucanase from B. macerans is more thermostable than the B. subtilis and B. amyloliquefaciens enzymes (Borriss, 1981; Borriss and Schroeder, 1981). However, at temperatures exceeding 65.degree. C. and at pH values of 4.5 to 5.5, which is typical for industrial mashing, the B. macerans .beta.-glucanase is rapidly inactivated. The B. macerans .beta.-glucanase gene has been cloned (Borriss et al., 1988) and its nucleotide sequence determined (Borriss et al., in prep.). Comparison of the derived amino acid sequence of B. macerans .beta.-glucanase with the derived sequences of B. subtilis and B. amyloliquefaciens .beta.-glucanases reveals an overall homology of 70%.
During recent years a number of attempts have been made to construct improved forms of existing, biologically active proteins to make them better suited for industrial processes and to widen their range of application. Much interest has been focused on increasing the thermostability of enzymes. It has been proposed that the thermostability of enzymes may be enhanced by single amino acid substitutions that decrease the entropy of unfolding (Matthews et al., 1987). Several tentative rules for increasing the thermostability of proteins have been established (Argos et al., 1979; Imanaka et al., 1986; Querol and Parilla, 1987) but precise predictions for changes of function as a consequence of changes in structure remain elusive.
Several researchers have conducted experiments with in vitro recombination of homologous genes giving rise to hybrid proteins retaining the biological activity of the parental molecules. Streuli et al. (1981) as well as Weck and coworkers (1981) constructed hybrid human leukocyte interferon genes. Some of the hybrid interferons extended the host cell range for protection against Vesicular Stomatitis and Encephalomyocarditis virus. Thus the AD hybrids combining portions of interferons A and D elicited significantly higher antiviral activities than either parental molecule in mouse L-929 cells, human He-La cells and primary rabbit kidney cells. Heat stability, pH stability and antigenic specificity were the same for the hybrid and parental interferon molecules.
Danish Patent Application 3368/87 discloses the combination of DNA sequences from the Bacillus licheniformis and Bacillus amyloliquefaciens .alpha.-amylase genes in order to obtain a chimeric .alpha.-amylase enzyme for the liquefaction of starch, which did not have a negative effect on the maximum percentage by weight of dextrose obtainable by saccharification with a glycoamylase. This negative effect was reduced with the chimeric .alpha.-amylase and the thermostable properties were retained in comparison with the parent enzymes.